Compounds and methods for PKCθ inhibition

ABSTRACT

The present invention provides a method of selectively inhibiting PKCθ in the presence of PKCδ, by administering to a subject in need thereof, a therapeutically effective amount of a compound of Formula I. The present invention also provides a method of inhibiting cytokine synthesis in a T cell, a method of inhibiting T cell proliferation, and a method of inhibiting the replication of and cytokine production by T lymphocytes, while not stimulating or inhibiting the replication of B lymphocytes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/561,759, filed Sep. 17, 2009, which claims priority to U.S.Provisional Application No. 61/098,640, filed Sep. 19, 2008, which areincorporated in their entirety herein for all purposes.

BACKGROUND OF THE INVENTION

T lymphocytes are an essential part of the immune response as they arenecessary for initiating the cellular response to pathogenic organisms,host cells that have become oncogenic and damaged tissue (seeFundamental Immunology, 6^(th) Edition, 2003, ed. Paul, Wm. LippencottWilliams & Wilkins). Functionally, T cells have been subdivided into twoclasses with distinct surface antigens, CD4 and CD8 and thisdifferentiation occurs in the Thymus. CD4+ cells are helper T cells andare further divided into Th1, Th2 and Th17 (see Tesmer, L. A., et al.(2008) Immunological Reviews, 228, 87-113, Harington, L. et al. (2005)Nat. Immunol. 6, 1123) which produce distinct levels of differentcytokines (see below). CD8+ cells are cytolytic cells and are involvedin lysis of tumors or viral infected cells. These responses are usuallyinitiated by the interaction of a specific surface protein of the Tcell, the T cell Receptor, TCR, (Davis, M. and Bjorkman, P. (1988)Nature 334, 395) with a “foreign” antigen on a defective host cell or onan Antigen Presenting Cell, APC, in the context of surface proteins ofthe Major Histocompatablilty Complex I or II (MHC I or II) and otherproteins in a complex structure, the Immune Synapse (Grakoui, A, et al.,(1999) Science 285,221). When the TCR interacts with a cognate antigen,it triggers a series of responses leading to activation of the T cell(Weiss, A. et al. (1991) Semin. Immulol. 3, 313, Weiss, A, (2003) Ann.Rev. Immunology). Activated T cells are induced to replicate morerapidly and, especially in the case of CD4+ cells, produce signalingproteins that are stimulatory (cytokines) or chemoattractive(chemokines) to inflammatory cells (i.e. lymphocytes, PMNLs, macrophageand monocytes) (Cook, D. N. (1996) J. Leukocyte Biol. 59, 61, Friedman,et al. (2006) Nat. Immunol. 7, 1101).

T cell activation can result in a disease process. In animal models Tcell responses described above have been shown to result in theinflammation of various tissues leading to experimental diseasesresembling, among others, asthma and COPD, rheumatoid arthritis,psoriasis, atopic dermatitis, uveitis, and multiple sclerosis. (Barnes,P. (2008) Nat. Rev. Immunol. 8,183; Martin (2003) Paediatric RespiratoryReviews, 5; S47). Furthermore, activated T cells and the relevantcytokines have been identified in the corresponding human diseases(Barnes, P (2008) Nat. Rev. Immunol. 8; 183; Krueger, J G and Bowcock(2005) Ann. Rev. Rheum. Dis. 64; 30, 13, 14). T cells are directlyactivated by foreign MHC I and II as well as other antigens ontransplanted tissues (liver, kidney, heart, etc.) resulting in graftrejection response which can be blocked by T cell specific antibodiesand/or by drugs known to block T cell activation (Odum, J. et al. (1993)Clin Nephrol. 39:230; Passerini, P. and Ponticelli, C. (2001) Curr OpinNephrol Hypertens. 10(2):189-93). T cell recruitment of macrophages isinvolved in pancreatic lesions leading to loss of (3-cells and Type 1diabetes (Cantor, J. and Haskins, K. (2006) Drug Discovery Today:Disease Mechanisms 3; 381). In addition, T cells can become oncogenic,resulting in T cell Lymphomas and Leukemias which in many instances arefatal (see Cheson, B. (2007) Sem. In Oncol. 34sup5; S3-S7).

Following the interaction of the T cell receptor, a series of kinasesand other enzymes is activated resulting in the transport to the nucleusand/or activation of the transcription factors NFkappaB, NFAT and AP1which causes transcription of cytokine and chemokine genes, as well asgenes involved in T cell replication, motility and survival(Cordronniere, N. et al. (2000) Proc. Nat. Acad. Sci. 97; 3394, Wulfing,C. and Davis, M. M. (1998) Science 282; 2266). The serine/threoninekinase PKC theta, is an essential step in this pathway (Sun et al.(2000) Nature 404:402-7, 19) and PKC theta deficient mice do not mount aT cell-driven inflammatory response (Healy, A. M. et al. (2006) J.Immunol. 177; 1886, Anderson, K. et al. (2006) Autoimmunity 39; 429).Human T cell lymphomas appear to have an upregulated PKCθ pathway(Vacca, A., et al. (2006) The EMBO Jour. 25, 1000) and mice deleted forPKCθ have reduced incidence of T cell lymphoma (Felli, P. M., et al.(2005) Oncogene 24; 992). Blocking the function of PKCθ may be a therapyfor several diseases in which T cells are involved.

It has also been shown that PKCθ activation in skeletal muscle may beinvolved in Type II diabetes (Li Y., et al. (2004) J. Biol. Chem. 279;45304), hence other diseases might be ameliorated by a inhibiting PKCθ.

The PKC family of serine/threonine kinases is comprised of at least 11members grouped into three subfamilies based on their cofactorrequirements: Conventional (alpha, beta1 and 2, gamma), novel (delta,epsilon, eta, theta) and atypical (xi, iota and zeta) (Newton, A. (2003)Biochem. J. 370; 361.), which are structurally similar, but arenecessary for many distinct cellular processes that are essential forcellular differentiation, survival and other specific cellularfunctions. For example, the novel PKC delta whose structure is mostsimilar to PKC theta, appears to be essential for regulating replicationof B lymphocytes (B cells) and reduced PKC delta causes uncontrolledexpansion of B cells leading to B cell invasion of tissues similar toSystemic Lupus Erythematosis (Mecklenbräuker, I. et al. (2002) Nature416; 860; Miyamoto, A., et al., (2002) Nature 416; 859). Conversely, theconventional PKC beta is necessary for B cell replication and survival(Saijo, vK., et al. (2003) Ann. N.Y. Acad. Sci. 987; 125). Hence,although it is apparent that blocking of PKC theta may be therapeuticfor diseases involving T cell activation, there is a need forisozyme-specific PKC theta inhibitors, in particular inhibitors ofPKC-theta that have minimal activity on PKC-delta and beta.Surprisingly, the present invention meets this, and other, needs.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method ofselectively inhibiting PKCθ in the presence of PKCδ, by administering toa subject in need thereof, a therapeutically effective amount of acompound of Formula I:

wherein X of Formula I is aryl or heteroaryl, each substituted with 1-5R¹ groups. Y of Formula I is —O—, —S(O)_(n)—, —N(R⁴)— and —C(R⁴)₂—,wherein subscript n is 0-2. Z of Formula I is —N═ or —CH═. Each R¹ ofFormula I is independently from the group consisting of H, halogen, C₁₋₈alkyl, C₁₋₆ heteroalkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₁₋₆ haloalkoxy, —OR^(1a), —C(O)R^(1a), —C(O)OR^(1a),—C(O)NR^(1a)R^(1b), —NR^(1a)R^(1b), —SR^(1a), —N(R^(1a))C(O)R^(1b),—N(R^(1a))C(O)OR^(1b), —N(R^(1a))C(O)NR^(1a)R^(1b), —OP(O)(OR^(1a))₂,—S(O)₂OR^(1a), —S(O)₂NR^(1a)R^(1b), —S(O)₂—C₁₋₆ haloalkyl, —CN,cycloalkyl, heterocycloalkyl, aryl or heteroaryl. Each of R^(1a) andR^(1b) of Formula I is independently H or C₁₋₆ alkyl. Each R² of FormulaI is independently H, halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, —NR^(1a)R^(1b), —NR^(1a)C(O)—C₁₋₆ alkyl, —NR^(1a)C(O)—C₁₋₆haloalkyl, —NR^(1a)—(CH₂)—NR^(1a)R^(1b), —NR^(1a)—C(O)—NR^(1a)R^(1b), or—NR^(1a)—C(O)OR^(1a), alternatively, adjacent R¹ groups and adjacent R²groups can be combined to form a cycloalkyl, heterocycloalkyl, aryl orheteroaryl. R³ of Formula I is —NR^(3a)R^(3b) or —NCO. Each of R^(3a)and R^(3b) of Formula I are independently H, C₁₋₆ alkyl, —C(O)—C₁₋₆alkyl, —C(O)—C₁₋₆ haloalkyl, —(CH₂)—NR^(1a)R^(1b), —C(O)—NR^(1a)R^(1b),—C(O)OR^(1a), —C(S)CN, an amino acid residue, a peptide or anoligopeptide. Each R⁴ of Formula I is independently H or C₁₋₆ alkyl, orwhen more than one R⁴ group is attached to the same atom, the R⁴ groupsare optionally combined to form a C₅₋₈ cycloalkyl. The compounds of thepresent invention also include the salts, hydrates and prodrugs thereof.In this manner, PKCθ is selectively inhibited in the presence of PKCδ.

In a second embodiment, the present invention provides a method ofinhibiting cytokine synthesis in a T cell, by administering to a subjectin need thereof, a therapeutically effective amount of a compound ofFormula I.

In a third embodiment, the present invention provides a method ofinhibiting T cell proliferation, by administering to a subject in needthereof, a therapeutically effective amount of a compound of Formula I.

In a fourth embodiment, the present invention provides a method ofinhibiting the replication of and cytokine production by T lymphocytes,while not stimulating or inhibiting the replication of B lymphocytes, byadministering to a subject in need thereof, a therapeutically effectiveamount of a compound of Formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of -4-(substituted phenoxy)-anilines (4).

FIG. 2 shows the synthesis of 4-(halogenated phenoxy)-anilines (8).

FIG. 3 shows the synthesis of 4-(substituted phenylthio)-anilines (12).

FIG. 4 shows the synthesis of 4-[substituted (phenylsulfinyl andphenylsulfonyl)]-anilines (13 and 14, respectively).

FIG. 5 shows the synthesis of N-acylated aniline-2,4-dichlorophenylether.

FIG. 6 shows the synthesis of N-(2,4-dichlorophenoxyphenyl)urea.

FIG. 7 shows the synthesis of N-acylated 4-(substitutedphenylthio)anilines ether and 4-(tert-butylphenylthio)acetanilide (26).

FIG. 8 shows the synthesis of N-acylated 4-[substituted (phenylsulfinyland phenylsulfonyl)]-anilines.

FIG. 9 shows the synthesis of4′-(tert-butylphenoxy)-2-dimethylaminoacetanilide.

FIG. 10 shows B-cells DNA synthesis was normal in the presence of PKCtheta inhibitor compounds.

FIG. 11 shows B-cell proliferation was “hyperstimulated” by a PKC deltainhibitor.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention is drawn to the selective inhibition of proteinkinase C-theta (PKCθ) in the presence of other isozymes such as PKCδ.Protein kinase C (PKC) phosphorylates other proteins, thereby alteringthe function of the protein. Isoforms of PKC include, but are notlimited to, α, βI, βII, γ, δ, ε, η, θ, ζ, ι and ξ. PKCθ is involved in Tcell activation, which is involved in the proliferation of a variety ofdiseases. Accordingly, inhibiting T cell activation caused by PKCθ isuseful for the treatment of a variety of diseases.

II. Definitions

As used herein, “administering” refers to oral administration,administration as a suppository, topical contact, parenteral,intravenous, intraperitoneal, intramuscular, intralesional, intranasalor subcutaneous administration, intrathecal administration, or theimplantation of a slow-release device e.g., a mini-osmotic pump, to thesubject.

As used herein, the term “subject” refers to animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. In certainembodiments, the subject is a human.

As used herein, the terms “therapeutically effective amount or dose” or“therapeutically sufficient amount or dose” or “effective or sufficientamount or dose” refer to a dose that produces therapeutic effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. For example, C₁-C₈ alkyl includes, but is not limited to,methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, neopentyl, t-pentyl, iso-pentyl, hexyl, heptyl,octyl, etc. In some embodiments, alkyl refers to C₁₋₆ alkyl.

As used herein, the term “heteroalkyl” refers to an alkyl group havingfrom 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms canalso be useful, including, but not limited to, B, Al, Si and P. Theheteroatoms can also be oxidized, such as, but not limited to, —S(O)—and —S(O)₂—. For example, heteroalkyl includes, but is not limited to,ethers, thioethers and alkyl-amines.

As used herein, the term “haloalkyl” refers to alkyl as defined abovewhere some or all of the hydrogen atoms are substituted with halogenatoms. Halogen (halo) preferably represents chloro or fluoro, but canalso be bromo or iodo. For example, haloalkyl includes trifluoromethyl,fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro”defines a compound or radical which has at least two available hydrogenssubstituted with fluorine. For example, perfluorophenyl refers to1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to1,1,1-trifluoromethyl, and perfluoromethoxy refers to1,1,1-trifluoromethoxy.

As used herein, the term “alkenyl” refers to either a straight chain orbranched hydrocarbon of 2 to 6 carbon atoms, having at least one doublebond. Examples of alkenyl groups include, but are not limited to, vinyl,propenyl, isopropenyl, butenyl, isobutenyl, butadienyl, pentenyl orhexadienyl.

As used herein, the term “alkynyl” refers to either a straight chain orbranched hydrocarbon of 2 to 6 carbon atoms, having at least one triplebond. Examples of alkynyl groups include, but are not limited to,acetylenyl, propynyl and butyryl.

As used herein, the term “alkoxy” refers to alkyl with the inclusion ofan oxygen atom, for example, methoxy, ethoxy, etc. “Haloalkoxy” is asdefined for alkoxy where some or all of the hydrogen atoms aresubstituted with halogen atoms. For example, haloalkoxy includestrifluoromethoxy, etc.

As used herein, the term “cycloalkyl” refers to a saturated or partiallyunsaturated, monocyclic, fused bicyclic or bridged polycyclic ringassembly containing from 3 to 12 ring atoms, or the number of atomsindicated For example, C₃₋₈cycloalkyl includes cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and up to cyclooctyl.

As used herein, the term “heterocycle” refers to a ring system havingfrom 3 ring members to about 20 ring members and from 1 to about 5heteroatoms such as N, O and S. Preferred heterocycles have 5-10 ringmembers and 1-4 heteroatoms. More preferred heterocycles have 5-6 ringmembers and 1-2 heteroatoms. Additional heteroatoms can also be useful,including, but not limited to, B, Al, Si and P. The heteroatoms can alsobe oxidized, such as, but not limited to, —S(O)— and —S(O)₂—. Forexample, heterocycle includes, but is not limited to, tetrahydrofuranyl,tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl,imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl,piperidinyl, indolinyl, quinuclidinyl and1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

As used herein, the term “aryl” refers to a monocyclic or fusedbicyclic, tricyclic or greater, aromatic ring assembly containing 6 to16 ring carbon atoms. For example, aryl may be phenyl, benzyl ornaphthyl, preferably phenyl. “Arylene” means a divalent radical derivedfrom an aryl group. Aryl groups can be mono-, di- or tri-substituted byone, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy,halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy andoxy-C₂-C₃-alkylene; all of which are optionally further substituted, forinstance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to twoadjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy.Oxy-C₂-C₃-alkylene is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. Anexample for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Preferred as aryl is naphthyl, phenyl or phenyl mono- or disubstitutedby alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenylor phenyl-mono- or disubstituted by alkoxy, halogen or trifluoromethyl,and in particular phenyl.

Examples of substituted phenyl groups as R are, e.g. 4-chlorophen-1-yl,3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methylphen-1-yl,4-aminomethylphen-1-yl, 4-methoxyethylaminomethylphen-1-yl,4-hydroxyethylaminomethylphen-1-yl,4-hydroxyethyl-(methyl)-aminomethylphen-1-yl, 3-aminomethylphen-1-yl,4-N-acetylaminomethylphen-1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl,2-aminophen-1-yl, 4-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phen-yl,4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl,4-(morpholin-1-ylmethyl)-phen-1-yl,4-(2-methoxyethylaminomethyl)-phen-1-yl and4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl,4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl, and4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally substitutedin the heterocyclic ring.

As used herein, the term “heteroaryl” refers to a monocyclic or fusedbicyclic or tricyclic aromatic ring assembly containing 5 to 16 ringatoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O orS. Preferred heteroaryls have 5-10 ring members and 1-4 heteroatoms.More preferred heteroaryls have 5-6 ring members and 1-2 heteroatoms.For example, heteroaryl includes pyridyl, indolyl, indazolyl,quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl,furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any otherradicals substituted, especially mono- or di-substituted, by e.g. alkyl,nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinylrepresents preferably 2-, 3- or 4-quinolinyl. Isoquinolinyl representspreferably 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranylrepresents preferably 3-benzopyranyl or 3-benzothiopyranyl,respectively. Thiazolyl represents preferably 2- or 4-thiazolyl, andmost preferred, 4-thiazolyl. Triazolyl is preferably 1-, 2- or5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl,thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl,thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl,benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted,especially mono- or di-substituted.

Substituents for the aryl and heteroaryl groups are varied and areselected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂,—CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′,—NR″C(O)₂R′—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

As used herein, the term “halogen” refers to fluorine, chlorine, bromineand iodine.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods of the present invention. Illustrativeexamples of pharmaceutically acceptable salts are mineral acid(hydrochloric acid, hydrobromic acid, phosphoric acid, and the like)salts, organic acid (acetic acid, propionic acid, glutamic acid, citricacid and the like) salts, quaternary ammonium (methyl iodide, ethyliodide, and the like) salts. It is understood that the pharmaceuticallyacceptable salts are non-toxic. Additional information on suitablepharmaceutically acceptable salts can be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, which is incorporated herein by reference.

As used herein, the term “hydrate” refers to a compound that iscomplexed to at least one water molecule. The compounds of the presentinvention can be complexed with from 1 to 10 water molecules.

As used herein, the term “prodrug” refers to covalently bonded carrierswhich are capable of releasing the active agent of the methods of thepresent invention, when the prodrug is administered to a mammaliansubject. Release of the active ingredient occurs in vivo. Prodrugs canbe prepared by techniques known to one skilled in the art. Thesetechniques generally modify appropriate functional groups in a givencompound. These modified functional groups however regenerate originalfunctional groups by routine manipulation or in vivo. Prodrugs of theactive agents of the present invention include active agents wherein ahydroxy, amidino, guanidino, amino, carboxylic or a similar group ismodified.

As used herein, the term “pharmaceutical excipient” refers to asubstance that aids the administration of an active agent to andabsorption by a subject. Pharmaceutical excipients useful in the presentinvention include, but are not limited to, binders, fillers,disintegrants, lubricants, coatings, sweeteners, flavors and colors. Oneof skill in the art will recognize that other pharmaceutical excipientsare useful in the present invention.

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Allthree terms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins, wherein the amino acid residues are linked bycovalent peptide bonds. In some instances, amino acid polymers havingmore than 50 amino acids are referred to as proteins.

As used herein, the term “oligopeptide” refers to a short polymer ofamino acid residues, about 2-15. Oligopeptide includes amino acidoligomers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid oligomers and non-naturallyoccurring amino acid oligomers.

As used here, the terms “inhibition”, “inhibits” and “inhibitor” referto a compound that prohibits or a method of prohibiting, a specificaction or function. “Selectively inhibiting” refers to the inhibition ofone species in the presence of another species that is not inhibited.For example, in the present invention, PKCθ is selectively inhibited inthe presence of PKCδ, among others.

As used here, the term “amino acid residue” refers to naturallyoccurring and synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function in a manner similar to the naturallyoccurring amino acids, that are linked to another moiety, such as viaformation of an amide bond. Naturally occurring amino acids are thoseencoded by the genetic code, as well as those amino acids that are latermodified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine.

“Amino acid analogs” refers to compounds that have the same basicchemical structure as a naturally occurring amino acid, i.e., an αcarbon that is bound to a hydrogen, a carboxyl group, an amino group,and an R group, e.g., homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid.

“Unnatural amino acids” are not encoded by the genetic code and can, butdo not necessarily have the same basic structure as a naturallyoccurring amino acid. Unnatural amino acids include, but are not limitedto azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid,tertiary-butylglycine, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,ornithine, pentylglycine, pipecolic acid and thioproline.

“Amino acid mimetics” refers to chemical compounds that have a structurethat is different from the general chemical structure of an amino acid,but that functions in a manner similar to a naturally occurring aminoacid.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

As used herein, the term “disease process” refers to a cellulardisturbance that gives rise to the symptoms defining a disease.

III. Compounds

The compounds of the present invention include any compound thatselectively inhibits PKCθ in the presence of PKCδ. In some embodiments,the compounds of the present invention include those of Formula I:

wherein X of Formula I is aryl or heteroaryl, each substituted with 1-5R¹ groups. Y of Formula I is —O—, —S(O)_(n)—, —N(R⁴)— and —C(R⁴)₂—,wherein subscript n is 0-2. Z of Formula I is —N═ or —CH═. Each R¹ ofFormula I is independently from the group consisting of H, halogen, C₁₋₈alkyl, C₁₋₆ heteroalkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₁₋₆ haloalkoxy, —OR^(1a)—C(O)R^(1a), —C(O)OR^(1a), —C(O)NR^(1a)R^(1b),—NR^(1a)R^(1b), —SR^(1a), —N(R^(1a))C(O)R^(1b), —N(R^(1a))C(O)OR^(1b),—N(R^(1a))C(O)NR^(1a)R^(1b), —OP(O)(OR^(1a))₂, —S(O)₂OR^(1a),—S(O)₂NR^(1a)R^(1b), —S(O)₂—C₁₋₆ haloalkyl, —CN, cycloalkyl,heterocycloalkyl, aryl or heteroaryl. Each of R^(1a) and R^(1b) ofFormula I is independently H or C₁₋₆ alkyl. Each R² of Formula I isindependently H, halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, —NR^(1a)R^(1b), —NR^(1a)C(O)—C₁₋₆ alkyl, —NR^(1a)C(O)—C₁₋₆haloalkyl, —NR^(1a)—(CH₂)—NR^(1a)R^(1b), —NR^(1a)—C(O)—NR^(1a)R^(1b), or—NR^(1a)—C(O)OR^(1a), alternatively, adjacent R¹ groups and adjacent R²groups can be combined to form a cycloalkyl, heterocycloalkyl, aryl orheteroaryl. R³ of Formula I is —NR^(3a)R^(3b) or —NCO. Each of R^(3a)and R^(3b) of Formula I are independently H, C₁₋₆ alkyl, —C(O)—C₁₋₆alkyl, —C(O)—C₁₋₆ haloalkyl, —(CH₂)—NR^(1a)R^(1b), —C(O)—NR^(1a)R^(1b),—C(O)OR^(1a), —C(S)CN, an amino acid residue, a peptide or anoligopeptide. Each R⁴ of Formula I is independently H or C₁₋₆ alkyl, orwhen more than one R⁴ group is attached to the same atom, the R⁴ groupsare optionally combined to form a C₅₋₈ cycloalkyl. The compounds of thepresent invention also include the salts, hydrates and prodrugs thereof.

In other embodiments, the compounds of the present are those of FormulaIa:

wherein each R¹ of Formula Ia is independently H, halogen, C₁₋₈ alkyl,C₁₋₆ heteroalkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆haloalkoxy, —OR^(1a), —CN, cycloalkyl, heterocycloalkyl, aryl orheteroaryl, and each of R^(3a) and R^(3b) of Formula Ia areindependently H, —C(O)—C₁₋₆ alkyl, an amino acid residue, a peptide oran oligopeptide. In still other embodiments, each R¹ of Formula Ia isindependently H, halogen, C₁₋₈ alkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy,—C(O)OR^(1a), cycloalkyl, or heteroaryl. Furthermore, each R² of FormulaIa is independently H, halogen, or —NR^(1a)C(O)—C₁₋₆ alkyl. In yet otherembodiments, each R¹ of Formula Ia is independently H, methyl, n-propyl,isopropyl, t-butyl, t-pentyl, Cl, Br, CF₃, OCF₃, cyclopentyl, pyrrolyl,or CO₂H, and each R² is independently H or Cl.

In another embodiment, R^(3a) of Formula I is an amino acid residue, andR^(3b) is H. In other embodiments, the amino acid residue is an arginineresidue.

In other embodiments, the compound has Formula Ib:

In some other embodiments, Y of Formula Ib is S. In still otherembodiments, Y of Formula Ib is O. In some embodiments, each R¹ ofFormula Ib is independently H, methyl, n-propyl, isopropyl, t-butyl,t-pentyl, Cl, Br, CF₃, OCF₃, cyclopentyl, pyrrolyl, or CO₂H. In yetother embodiments, each R¹ of Formula Ib is independently C₁₋₈ alkyl orcycloalkyl. In still yet other embodiments, each R¹ of Formula Ib isindependently 4-t-butyl, 4-cyclopentyl or 4-t-pentyl.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

The compounds of the present invention can be prepared by a variety ofmethods known to one of skill in the art, such as shown in FIGS. 1-9.One method of preparing the compounds of the present invention involvescondensation of a para-fluoro-nitrobenzene with an alcohol, followed byreduction of the nitro group to an amine (FIGS. 1 and 2). A variety ofalcohols can be used, such as phenols, heteroaryl alcohols and alkanols,including cycloalkanols. The nitro group can be reduced by a variety ofreagents known to one of skill in the art, including, but not limitedto, Pd/C and Fe/NH₄Cl. In addition to the aryl-nitro starting materialshown in FIG. 1, heteroaryl-nitro compounds can also be used in thecondensation method to prepare compounds of the present invention. Oneof skill in the art will appreciate that other alcohols and reductionsteps are useful in preparing the compounds of the present invention.

The condensation method can also be used with thiols to prepare adisulfide (FIG. 3). The nitro group can be reduced as previouslydescribed. Thiols useful in the condensation method for making compoundsof the present invention include, but are not limited to, thiophenols.The disulfide can also be oxidized to the sulfoxide and the sulfoneusing any oxidizing agent known to one of skill in the art, including,but not limited to, hydrogen peroxide (FIGS. 4 and 8). One of skill inthe art will appreciate that other oxidizing agents are useful in thepresent invention.

The amine group of the compounds of the present invention can bederivatized by a variety of methods known to one of skill in the art. Insome embodiments, the amine can be acylated such as with an anhydride(FIGS. 5 and 7) or by condensation with a carboxylic acid (FIG. 9). Inother embodiments, the amine can be reacted with an isocyanate to affordthe urea (FIG. 6). Additional methods of making the compounds of thepresent invention are known to one of skill in the art, for example,those described in Comprehensive Organic Transformations, 2d ed.,Richard C. Larock, 1999, and methods described in U.S. Pat. No.4,130,433, incorporated in its entirety herein. The starting materialsfor the methods described above are commercially available(Sigma-Aldrich) or can be prepared by methods known to one of skill inthe art.

The starting materials used in the synthetic methods described above canbe substituted or unsubstituted. Substituents for starting materialsinclude, but are not limited to, -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′,—R′, —CN, —NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′,—NR″C(O)₂R′—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, cycloalkyl,heterocycloalkyl, aryl and heteroaryl, where R′, R″ and R′″ areindependently selected from hydrogen and (C₁-C₈)alkyl.

One of skill in the art will appreciate that certain compounds of thepresent invention possess asymmetric carbon atoms (optical centers) ordouble bonds; the racemates, diastereomers, geometric isomers andindividual isomers are all intended to be encompassed within the scopeof the present invention.

IV. Methods of Identifying Compounds as Selectively Inhibiting PKCθ inthe Presence of PKCδ

Using the XenoGene™ system described within, compounds inhibiting PKCθare identified. The XenoGene™ system is a technology developed andpatented by CompleGen wherein genes (“XenoGenes”™) from a targetorganism (e.g. humans) are used to functionally replace essential genesof a simple organism (yeast). These modified yeast are used to selectcompounds that act specifically on the protein encoded by the targetgene and eliminate compounds that are not specific. XenoGene™ constructsprovide a very high throughput screening system that allowsidentification of compounds active on the function of the target proteinin a cell under physiological conditions on “first-pass” screening andfurther, to selectively counter-screen against 200 human targets or moreif necessary. The “read out” of the XenoGene™ assay is simply growth(inactive compound) or no growth (active compound) of the yeastcontaining the target gene, and growth of control strains that do notcontain the target gene. The relative potencies of the active compoundsis determined by titration to find the concentration of compoundproviding 50% inhibition of growth (IC₅₀) against the yeast containingthe target gene (Table 1).

TABLE I IC₅₀ Values for Compounds of Formula Ia (Ia)

Compound R¹ R² R^(3a) R^(3b) Y Z IC₅₀ ¹ 471 4-t-butyl H H H O —CH═ +++486 4-t-butyl H H H O —N═ + 498 2-Cl, 4-OCF₃ H H H O —CH═ +++ 499 2-Cl,4-CF₃ H H H O —CH═ +++ 500 H H H H O —CH═ + 501 2,4-Cl₂ H C(O)Me H O—CH═ + 503 2-Cl, 4-OCF₃ 3-Cl H H O —CH═ ++ 504 3,5-Cl₂ H H H O —CH═ ++505 4-Cl, 3-CH₃ H H H O —CH═ +++ 506 2,4-Cl₂ H H H O —CH═ ++ 507 4-Cl,3-CH₃ H H H O —CH═ ++ 508 4-CH₃ H H H O —CH═ ++ 509 3,5-Cl₂ H H H O —CH═++ 510 4-OCF₃ H H H O —CH═ +++ 553 2-COOH H H H O —CH═ + 730 4-t-pentylH H H O —CH═ +++ 733 4-CF₃ H H H O —CH═ ++ 740 4-t-butyl H H H S —CH═+++ 741 4-CF₃ H H H S —CH═ +++ 742 4-isopropyl H H H O —CH═ +++ 7454-n-propyl H H H O —CH═ +++ 747 2-Br, 4-CF₃ H H H O —CH═ +++ 7494-t-butyl H H H SO₂ —CH═ ++ 750 4-t-butyl H H H SO₂ —CH═ + 751 4-CF₃ H HH SO —CH═ + 753 4-CF₃ H H H SO₂ —CH═ ++ 761 4-cyclopentyl H H H O —CH═+++ 762 1-pyrrolyl H H H O —CH═ ++ ¹+++, <1 μM; ++, 1-10 μM; +, >10 μM.

To identify compounds as being selective PKC theta inhibitors the IC₅₀values are determined against XenoGene™ strains containing otherXenoGenes™, especially certain other PKC genes (see Example 10). One ofskill in the will appreciate that other compounds of the presentinvention selectively inhibit PKCθ in the presence of PKCδ.

V. Method of Selectively Inhibiting PKCθ in the Presence of PKCδ

In some embodiments, the present invention provides a method ofselectively inhibiting PKCθ in the presence of PKCδ, by administering toa subject in need thereof, a therapeutically effective amount of acompound of Formula I:

A. Formulations

The compounds of the present invention can be formulated in a variety ofdifferent manners known to one of skill in the art. Pharmaceuticallyacceptable carriers are determined in part by the particular compositionbeing administered, as well as by the particular method used toadminister the composition. Accordingly, there are a wide variety ofsuitable formulations of pharmaceutical compositions of the presentinvention (see, e.g., Remington's Pharmaceutical Sciences, 20^(th) ed.,2003). Effective formulations include oral and nasal formulations,formulations for parenteral administration, and compositions formulatedfor with extended release.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of a compound of the presentinvention suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets, depots or tablets, each containing a predeterminedamount of the active ingredient, as liquids, solids, granules orgelatin; (c) suspensions in an appropriate liquid; (d) suitableemulsions; and (e) patches. The liquid solutions described above can besterile solutions. The pharmaceutical forms can include one or more oflactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch,potato starch, microcrystalline cellulose, gelatin, colloidal silicondioxide, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, e.g., sucrose, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, carriers known in the art.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents. Preferred pharmaceutical preparations candeliver the compounds of the invention in a sustained releaseformulation.

Pharmaceutical preparations useful in the present invention also includeextended-release formulations. In some embodiments, extended-releaseformulations useful in the present invention are described in U.S. Pat.No. 6,699,508, which can be prepared according to U.S. Pat. No.7,125,567, both patents incorporated herein by reference.

The pharmaceutical preparations are typically delivered to a mammal,including humans and non-human mammals. Non-human mammals treated usingthe present methods include domesticated animals (i.e., canine, feline,murine, rodentia, and lagomorpha) and agricultural animals (bovine,equine, ovine, porcine).

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents.

B. Administration

The compounds of the present invention can be administered as frequentlyas necessary, including hourly, daily, weekly or monthly. The compoundsutilized in the pharmaceutical method of the invention are administeredat the initial dosage of about 0.0001 mg/kg to about 1000 mg/kg daily. Adaily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may bevaried depending upon the requirements of the patient, the severity ofthe condition being treated, and the compound being employed. Forexample, dosages can be empirically determined considering the type andstage of disease diagnosed in a particular patient. The doseadministered to a patient, in the context of the present inventionshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular compound in a particular patient.Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired. Doses can be given daily, or on alternate days, asdetermined by the treating physician. Doses can also be given on aregular or continuous basis over longer periods of time (weeks, monthsor years), such as through the use of a subdermal capsule, sachet ordepot, or via a patch or pump.

The pharmaceutical compositions can be administered to the patient in avariety of ways, including topically, parenterally, intravenously,intradermally, subcutaneously, intramuscularly, colonically, rectally orintraperitoneally. Preferably, the pharmaceutical compositions areadministered parenterally, topically, intravenously, intramuscularly,subcutaneously, orally, or nasally, such as via inhalation.

In practicing the methods of the present invention, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents. The additional drugs used in the combinationprotocols of the present invention can be administered separately or oneor more of the drugs used in the combination protocols can beadministered together, such as in an admixture. Where one or more drugsare administered separately, the timing and schedule of administrationof each drug can vary. The other therapeutic or diagnostic agents can beadministered at the same time as the compounds of the present invention,separately or at different times.

C. Treatment of Diseases

The method of the present invention can be used to treat a variety ofdiseases and conditions involving PKCθ, including, but not limited to,inflammatory diseases (uveitis, psoriasis, rheumatoid arthritis,multiple sclerosis, inflammatory bowel syndrome, Crohn's disease),atopic dermatitis, prevention of organ transplant rejection, T celllymphomas and leukemia, diseases involving degranulation of basophilicgranulocytes and reversal of insulin resistant type II diabetes.

VI. Inhibition of the Activation of Effector T Cells

In some embodiments, the present invention provides a method ofinhibiting cytokine synthesis in a T cell, by administering to a subjectin need thereof, a therapeutically effective amount of a compound ofFormula I. In other embodiments, the compound is of Formula Ib. In someother embodiments, X of Formula Ib is S. In still other embodiments, Xof Formula Ib is O.

In other embodiments, the present invention provides a method ofinhibiting T cell proliferation, by administering to a subject in needthereof, a therapeutically effective amount of a compound of Formula I.In some embodiments, the compound is of Formula Ib. In some otherembodiments, X of Formula Ib is S. In still other embodiments, X ofFormula Ib is O.

In some other embodiments, the present invention provides a method ofinhibiting the replication of and cytokine production by T lymphocytes,while not stimulating or inhibiting the replication of B lymphocytes, byadministering to a subject in need thereof, a therapeutically effectiveamount of a compound of Formula I. In other embodiments, the compound isof Formula Ib. In some other embodiments, X of Formula Ib is S. In stillother embodiments, X of Formula Ib is O.

Compounds useful in the method of inhibiting cytokine synthesis in a Tcell or inhibiting T cell proliferation are identified by their abilityto inhibit antigen-specific T cell activation. Assays for inhibitingantigen-specific T cell activation are known to one of skill in the art.In some embodiments, the assay involves immunizing mice with ovalbumin,isolating mouse splenic T cells and incubating the isolated cells withmitomycin C treated, unimmunized mouse spleen cells or T cell depletedspleen cells. Proliferation was measured by incorporation of ³H-TdR (seeExample 11). One of skill in the art will appreciate that other assaysare useful in the present invention.

The compounds of the present invention can also be screened for theability to inhibit T cell lymphomas. Assays for inhibiting T celllymphomas are known to one of skill in the art. In some embodiments, theassay involves mice generated from the nucleus of a mature T cell with a“fixed” rearranged T cell receptor. These T cells spontaneously generateT cell lymphomas which resemble human T cell lymphomas. T cell lymphomasisolated from the mice can then be treated with compounds of the presentinvention. After sufficient time to allow for cell growth, live cellsare counted by flow cytometry (Serwold et al. (2007) J. Immunol. 179;928). One of skill in the art will appreciate that other assays areuseful in the present invention.

VII. Examples Example 1 Synthesis of 4-(substituted phenoxy)-anilines(4)

In a 5 mL microwave tube, 200 mg of the substituted phenol (1) and 1equiv. of 1-fluoro-4-nitrobenzene (2) were dissolved in 4 mL of DMF and2.9 equiv. of Na₂CO₃ was added. The reaction mixture was heated in themicrowave at 170° C. for 1 h. The mixture was extracted with EtOAc andwater; the organic phase was washed with brine and dried over MgSO₄. TheEtOAc was evaporated on the rotary evaporator to give the crude product(3). (60-70% yield).

In a hydrogenation bottle, crude (3) was dissolved in MeOH; solution waspurged with N₂ prior to the addition of 50-100 mg of 5 wt % Pd onactivated carbon. The reaction mixture was placed in a hydrogenator andshaken with 1-2 atm H₂ for 1-2 hr. Upon completion of the reaction, thecatalyst was filtered through the fritted funnel and was rinsed withMeOH. MeOH was evaporated using the rotary evaporator to give the finalcrude product (4). (Approx. 80% yield).

Example 2 Synthesis of 4-(halogenated phenoxy)-anilines (8)

In a 5 mL microwave tube, 200 mg of the substituted phenol (5) and 1equiv. of 1-fluoro-4-nitrobenzene (6) were dissolved in 4 mL of DMF,then 2.9 equiv. of Na₂CO₃ was added. The reaction mixture was allowed toheat in the microwave at 170° C. for 1 h. The mixture was partitionedbetween EtOAc and water; the organic phase was washed with brine andthen dried over MgSO₄. The EtOAc was evaporated by the rotary evaporatorto give the crude product (7). (Approx. 70-90% yield).

In a round bottom flask, crude (7) was dissolved in 50 L of EtOH; 15equiv. of Fe powder and 25 mL of saturated aqueous NH₄Cl were added tothe solution mixture. The reaction mixture was allowed to reflux at 90°C. for 2 h. Fe powder was filtered from the reaction mixture with afritted funnel. The filtrate was concentrated using the rotaryevaporator. Crude product was extracted with EtOAc and water; theorganic phase was washed with brine and then dried over MgSO₄. Thesolvent was removed on the rotary evaporator, giving approximately65-85% yield of crude product (8).

Example 3 Synthesis of 4-(substituted phenylthio)-anilines (12)

In a 5 mL microwave tube, 200 mg of the substituted thiol (9) and 1equiv. of 1-fluoro-4-nitrobenzene (10) were dissolved in 4 mL of DMF,then 2.9 equiv. of Na₂CO₃ was added. The reaction mixture was heated inthe microwave at 170° C. for 1 h. The mixture was partitioned betweenEtOAc and water; the organic phase was washed with brine and then driedover MgSO₄. EtOAc was evaporated on the rotary evaporator to give thecrude product (11). (60-70% yield).

In a hydrogenation bottle, crude (11) was dissolved in MeOH; solutionwas purged with N₂ prior to the addition of 50-100 mg of 5 wt % Pd onactivated carbon. The reaction mixture was hydrogenated for 1 hr;reaction course monitored by LC/MS. Upon completion of the reaction, Pdwas filtered through a fitted funnel and was rinsed with MeOH. MeOH wasevaporated on the rotary evaporator to give the final crude product(12). (40-50% yield).

Example 4 Synthesis of 4-[substituted (phenylsulfinyl andphenylsulfonyl)]-anilines (13 and 14, respectively)

In a round bottom flask, crude (12) was dissolved in 1 mL of DMF. Excessof 30% H₂O₂ solution (approx. 3-5 mL) was subsequently added to thesolution. The reaction mixture was allowed to sit at room temperaturefor 24 h; reaction course was monitored by LC/MS. Reaction mixture wasconcentrated by the rotary evaporator to give the final crude sulfoxide(13).

Sulfone was obtained by the addition of excess of 30% H₂O₂ solution(another 3-5 mL). The reaction mixture was allowed to sit at R.T. for 72h to afford the final product (14). Reaction course was monitored byLC/MS.

Example 5 Synthesis of N-acylated aniline-2,4-dichlorophenyl ether

In a 20 mL vial, 4-(2,4-dichlorophenoxy)aniline (19) and 1 equiv. ofacetic anhydride (20) were dissolved in 3 mL of dichloromethane,followed by the addition of 1 equiv. of diisopropylethylamine. Thereaction mixture was allowed to stir at R.T. for 2 h. dichloro methane(DCM) was then evaporated by the rotary evaporator to give the desiredproduct (21).

Example 6 Synthesis of N-(2,4-dichlorophenoxyphenyl)urea

In a 20 mL vial, aniline-2,4-dichlorophenyl ether (19, 506) wasdissolved in 3 mL of dichloromethane and 2 equiv. of trimethylsilylisocyanate (22) was added. The reaction mixture was allowed to stir at40° C. for 48 h; reaction course was monitored by LC/MS. Precipitateswere filtered through the fitted funnel, washed with DCM several times,and then air-dried to afford the desired pure product (23). ExactMass=296; observed mass=296.7 (28% yield).

Example 7 Synthesis of N-acylated 4-(substituted phenylthio)anilinesether. 4-(tert-butylphenylthio)acetanilide (26)

In a 20 mL vial, 12 was dissolved in 5 mL of DCM. 1 equiv. of aceticanhydride (25) and 1 equiv. of DIEA were added. The reaction mixture wasallowed to stir at 40° C. for 24 h. Solvent was removed by the rotaryevaporator to afford (26).

Example 8 Synthesis of N-acylated 4-[substituted (phenylsulfinyl andphenylsulfonyl)]-anilines

In a 50 mL round bottom flask, 26 was dissolved in 1 mL of DMF prior tosubsequent addition of 3-5 mL of 30% H₂O₂ solution. The reaction wasallowed to stir at R.T. for 48 h, then at 35° C. for 4 h. Crude productwas extracted with EtOAc and water; organic phase was washed with brineand dried over MgSO₄. EtOAc was evaporated using the rotary evaporatorto give 27.

Example 9 Synthesis of 4′ tert-butylphenoxy)-2-dimethylaminoacetanilide

In a 50 mL round bottom flask, (28) and 1 equiv. of N,N-dimethylglycine(29) were dissolved in 5 mL of DMF, followed by the addition of 3 equiv.of diisopropylethylamine and 1.3 equiv. of HATU(0-(7-Azabenzotriazole-1-yl)-N,N,N′N′-tetramethyluroniumhexafluorophosphate). The reaction mixture was stirred at R.T. for 48 h.The mixture was diluted with EtOAc and crude product was extracted with5% NaHCO₃ solution twice. Organic phase was washed with brine and thendried over MgSO₄. EtOAc was evaporated using the rotary evaporator toafford (30). (30% yield).

Example 10 Identification of Compounds with Selective PKCθ InhibitionActivity

Human PKC theta gene (cDNA) was used to generate a XenoGene™ system aspreviously described (U.S. Pat. Nos. 6,998,261, 6,232,074), which wasused to screen compounds in a chemical compound library. XenoGene™systems used as counter-screens were constructed similarly using thegene for the appropriate counter-screen target. In particular, humangenes (cDNA) encoding PKC delta, PKC eta and PKC epsilon were used togenerate XenoGene™ systems.

Using the XenoGene™ system, the following compounds were identified asbeing selective PKC theta inhibitors.

PKC-theta Selectivity for PKC-theta v.¹ Target/ IC₅₀ PKC- PKC- PKC-yeast Compound (μM) delta epsilon eta PKC1 471 0.14 2.5 1.1 0.36 28 5041.6 8.1 8.8 1.8 505 0.40 13 11 3.0 506 1.6 1.9 2.5 0.63 9.4 740 0.05 2612 3.0 80 741 0.25 8.0 16 4.0 32 ¹These values are calculated by takingthe inverse of the ratio of (PKC-theta IC₅₀)/(PKC-X IC₅₀).

Example 11 Inhibition of Effector T Cells

The compounds of the present invention were tested for their ability toinhibit the activation of effector T cells. The procedures described arefamiliar to scientists skilled in immunological research and referencesare given as examples, but, are not meant to be limiting.

Inhibition of Antigen-Specific (Effector) T Cell Activation (CellularProliferation and Cytokine Production).

Mice were immunized i.p at least 2 weeks in advance with 100 mg solubleOVA (ovalbumin, Sigma) emulsified 1:1 in CFA (complete Freund'sadjuvant, Sigma). Mouse splenic T cells were isolated by magneticnegative selection (Miltenyi®, Untouched T Cell Isolation Kit) or FACSusing anti-CD3-PE-Cy7. 105 isolated T cells were incubated in microtiterwells with 105 mitomycin C treated, unimmunized mouse spleen cells or Tcell depleted spleen cells as Antigen Presenting Cells (APC) and 1 mgsoluble OVA in 100 microliters 10% FBS-RPMI for five days. Results weresimilar with mitomycin C-treated total spleen cells and mitomycinC-treated, T depleted spleen cells. Proliferation was measured byincorporation of ³H-TdR, added for the final 18 hrs of culture. Themitomycin C treated spleen cells or T-depleted spleen cells consistentlyyielded only background levels of ³H-TdR. Mononuclear spleen cells wereisolated from Mice (See Dubey, C. et al. (1995) J. Immunol. 155; 45). Tcell proliferation was measured by ³H-TdR incorporation. Cytokineproduction was measured by the CBA® multiplexed assay system (BDBiosciences) according to manufacturer's directions.

Inhibition of naïve T cell activation by compound 471. Mouse splenic Tcells were isolated from unimmunized mice by magnetic negative selection(Miltenyi®, Untouched T Cell Isolation Kit) or FACS usinganti-CD3-PE-Cy7. Isolated T cells were incubated in microtiter wellscoated with anti-CD3 monoclonal antibody and soluble anti-CD28monoclonal antibody, 10⁵ cells/100 μl in 10% FBS-RPMI as above, forthree days. Proliferation was measured by ³H-TdR incorporation.

Compound ³H-TdR cpm   0 31571 0.5 uM 21928 1.0 uM 880

Inhibition of T Cell Lymphomas.

Mice generated from the nucleus of a mature T cell (see Hochedlinger K,Jaenisch R. (2002) Nature 415; 1035) with a “fixed” rearranged T cellreceptor spontaneously generate T cell lymphomas which resemble human Tcell lymphomas. Several T cell lymphomas (1-4) isolated from these micewere treated with compound 471. All T cell lymphomas were inhibited bycompound 471. A B-cell hybridoma served as a non-T cell control and wasnot affected by the compound. Cells were seeded into 96-well plates(5,000 cells/well) in the presence of compound 471 at concentrationsranging from 0.1 to 5 uM. After 4 days growth (37° C., 5% CO₂), livecells were counted by flow cytometry (Serwold et al. (2007) J. Immunol.179; 928)

Cell Lym- Lym- B-cell Type Lymphoma 1 phoma 2 Lymphoma 4 phoma 5Hybridoma EC₅₀ 0.7 uM 0.2 uM <0.1 uM 0.7 uM >5 uM

Lack of Activity on B-Lymphocytes.

Mouse splenic B cells were isolated by negative magnetic selection(Miltenyi®, Untouched T Cell Isolation Kit) or FACS using anti-CD19-PE.Isolated B cells were incubated in microtiter wells with solubleanti-IgM and anti-CD40 monoclonal antibodies, 10⁵ cells/100 μl in 10%FBS-RPMI as above, for three days. Proliferation was measured by ³H-TdRincorporation.

As shown in FIG. 10, B-cells DNA synthesis was normal in the presence ofPKC theta inhibitor compounds. As shown in FIG. 11, B-cell proliferationwas “hyperstimulated” by a PKC delta inhibitor.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A method of selectively inhibiting PKCθ in thepresence of PKCδ, comprising the step of contacting a T-cell containingboth PKCθ and PKCδ, with an effective amount of a compound of FormulaIb:

wherein Y is selected from the group consisting of —O— and —S—; each R¹is independently selected from the group consisting of n-propyl,isopropyl, t-butyl, t-pentyl, CF₃, OCF₃, cyclopentyl, pyrrolyl, and CO₂Hand salts, hydrates and prodrugs thereof, thereby selectively inhibitingPKCθ.
 2. The method of claim 1, wherein Y is S.
 3. The method of claim1, wherein Y is O.
 4. The method of claim 1, wherein each R¹ isindependently selected from the group consisting of 4-t-butyl,4-cyclopentyl and 4-t-pentyl.
 5. The method of claim 1, wherein thecontacting is in vivo.
 6. The method of claim 1, wherein the contactingis ex vivo.
 7. The method of claim 1, wherein the compound is


8. The method of claim 1, wherein the contacting is ex vivo; and thecompound is